The present invention pertains in general to substrate formulations for enzyme assays, and, in particular, to substrate formulations for alkaline phosphatase assays which include a phosphated indigo cogener and a tetrazolium salt.
Assays for the detection of substances present in small amounts rely upon the use of indicators. In biological assays, one type of indicator involves the use of an enzyme attached to a probe which preferentially binds to a target substance to be detected. Once bound to the target, the enzyme may function as a reporter group by catalyzing a reaction with a substrate to produce a product which is readily detectable by unaided vision or by a machine.
One example of an enzyme which is capable of serving as a reporter group is alkaline phosphatase. The term alkaline phosphatase generally refers to a group of non-specific enzymes which hydrolyze monophosphoric esters over a pH range of approximately 8.5 to 10.5.
Although a number of substrates may be reacted with an alkaline phosphatase enzyme, reaction with an indigo cogener, preferably a 3-indolyl phophate salt such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP), produces a particularly useful signal. Alkaline phosphatase degrades BCIP into an organic moiety and a phosphate. The organic moieties dimerize to form a blue-colored product. A transphosphorylating buffer removes the phosphate transferred from the indigo cogener by the enzyme, thus avoiding accumulation of phosphate by the enzyme and consequent enzyme inhibition. Transphosphorylating buffers useful in such reactions include 2-amino-2-methyl-1-propanol (2A2M1P), diethanolamine, ethane amino ethanol, and 2-amino-2-methyl-1,3-propandiol. See Starkweather, U.S. Pat. No. 4,030,995; and U. K. Patent No. 1,263,202.
Enhancement of the alkaline phosphatase reaction with BCIP may be achieved by the addition of nitro blue tetrazolium (NBT). The products of the reaction of alkaline phosphatase with BCIP reduce NBT to insoluble blue diformazan, which may produce a stronger signal than is produced by the BCIP reaction products alone. See, e.g., Parent et al., Phytoprotection, 66, 53-57 (1985). However, a precipitate forms in an aqueous substrate formulation containing BCIP and NBT with about 24 hours of preparation. Formation of this precipitate is associated with a loss of activity, necessitating the separate packaging of NBT, BCIP and buffer in kits for the preparation of alkaline phosphatase subtrate formulations.